Process for reduction of organic disulphides



.ing in poor yields.

sort to high pressure or 2,820,780 PRGCESS FOR REDUCTIQN OF ORGANICDlSULP ll l Marcia Gutcho and Louis Laufer, Bronx, N. Y., assignors, bymesne assignments, to the United States of America as represented by theSecretary of the Navy No Drawing. Application Gotcha-30, 1953 Serial No.389,470

Claims. (Cl. 260-112) The present invention is concerned with thereduction of organic disulfide compounds of the general class RSSR'where R and R may be amino acid, peptide,

.alkyl or aryl group or other organic radicalto their respective thiols,RSH and R'SH.

As conducive to a clear understanding of the invention, it is noted thatdisulfides of amino acids and peptides such as cystine, diglutatruone,.diglutamylcystine, cystinyl diglycine and related cystine peptides maybe re- .duced by cyanide, or sodium amalgam but these reactions areincomplete and form undesirable by-products result- Alltyl and aryldisulfides are reduced to respective thiols by hydrogen in the presenceof metallic sulfides, but only under conditions of high pressure andelevated temperature.

It is accordingly among the objects of the invention to provide aprocess for the reduction of organic .disulfides to their respectivethiol forms rapidly and in good yield by the use of inexpensive reagentsand Without rehigh temperatures.

Another object is to provide a method wherebydiSulfides may beeliminated from mixtures containing them by converting such disulfidesto their respective thiol forms so that they may be removed .morereadily .from such mixtures as insoluble metal mercaptides.

A further object is to accomplish reduction of organic disulfide underconditions of pH and temperature which are conducive to stability of thefree thiol thus formed, such thiols being generally more resistant toreoxidation to disulfides at acidic pH (below 7 and preferably below5.0) and at low temperatures.

For exmple, dithioethanolamine CH2CE2SSCH:CHg

NHz N112 is highly toxic, but the reduced thiol OH OHr-SH has beenreported to have useful therapeutic properties. Also, many heatsensitive, enzymatically active proteins are inactivated by theformation of disulfides and may be reactivated by reducing the 8-8linkages essential to enzyme activity to the free SH (thiol) form attemperatures below 60 C.

In the refining of petroleum products, it is frequently necessary toremove aliphatic and aromatic disulfides to avoid poisoning of catalystsused in subsequent processing. Furthermore, sulfur compounds may producecorrosive decomposition products when present in motor fuels orlubricants. As a step in removal of disulfides from petroleum mixtures,it is convenient first to reduce them to free thiols so that they may beremoved more readily as insoluble metal mercaptides.

In the preparation of sulfhydryl amino acids and peptides which areuseful in nutrition and medical treatment, such as cysteine andglutathione, yields are frequently markedly reduced by formation ofdisulfides during proc- 'essing, which is difficult to avoid.

catalyst, the reactionwith H 8.

2,820,780 Fatented Jan. 21, 1958 ice The invention provides a means ofconverting such disulfides to the desired and products in the thiol formabove referred to.

The invention from one important aspect thereof, utilizes the surprisingdiscovery that metallic sulfides, particularly those of bismuth, leadand mercury, or other acid insoluble metal sulfide of molecular weightgreater than 60, catalyze a reaction between hydrogen sulfide andorganic disulfides in an aqueous'medium which reaction results in.rapidreductionof such disulfides to their respective thioliforms. Thisreaction proceeds whether the metallic sulfide is formed in situ or isprepared in advance and added to the reaction mixture, and is operativeat-substantially atmospheric pressure and temperatures below 100 .C. Inthe absence of such metal sulfide proceeds if at all at rates notindustrially practical.

In one embodiment of the invention, water-soluble salts of lead,mercury'or bismuth are added to aqueous solutions of peptide disulfidessuch as cystine, diglutamylcystine, cystinyldi'glycine or the disulfideform of glutathione, with or without addition of acid, and H 5 is passedthrough the solution at room temperature and atmospheric pressure. Theinsoluble metal sulfide is formed promptly in situ, and promptly andprogressively elfects formation of the respective thiol forms in thereaction mixture as gassing continues.

In another embodiment of this invention insoluble metal sulfide catalystisprepared by passing H 5 through a solution of a soluble salt of thedesired metal causing "precipitation of .thelinsoluble metal. sulfidewhichis subsequently dispersed into :thereaction mixture.

Disulfides which' form salts of the metals lead, mercury or bismuth,such as amino acids and peptides, may :be

converte'd,first to.such metal salts andthen treated either in solutionor suspension with H 8, thus carrying out in situ the decomposition ofsuchtmetal salt andthe for- 'mation of the metalsulfidewhichcthereuponcatalyzes the reduction of the free disulfide to thethiolzform.

The concentration of disulfide is not critical, being largely aimatterof convenience .and depending .upon the solubility of the disulfidecompound .or .compounds in the reaction mixture. lnthe aqueous systemsused, concentrations from 2 to 30% are preferred. Inrnixtures where thedesired end is the removal of undesirable disulfide components, theconcentrations of such compounds may be as little as 0.1%.

In aqueous-systems acidification to a pH below '5 is desirable becausethe metal sulfides generally precipitate more elfectively and thiols areless susceptible to reoxidation in subsequent processing if the mediumis acid.

The molar ratio of metal sulfide to disulfide present may be from 0.01to 1, preferably 0.01 to 0.1.

The H S is added most conveniently by diffusing the gas under thesurface of the reaction mixture at a rate just sufficient to maintainthe mixture saturated in respect to H 5 and provide a slight excess.

While the foregoing description is believed adequate to meet statutoryrequirements, a few specific examples will nevertheless now be setforth.

Example I To 10 parts by weight of a 6% aqueous solution of oxidizedglutathione (GSSG) having a natural pH of about 2.5, is added 1 part ofa 10% solution of lead acetate tri-hydrate Pb(C l-l O) .3H O. Acapillary gas diffusing tube is inserted into the liquid and H 8 passedinto the solution. After sixty minutes, assay of the reaction mixtureindicates that of the GSSG has been reduced to GSH. After removal of thePbS which precipitates on gassing, the GSH may be recovered from thesolution as its insoluble cuprous salt.

-. converted to cysteine through a which in order of 1% Example II H 8gas is passed through a mercuric chloride solution in which the mercurysalt has been brought into solution by addition ofsufiicientconcentrated' HCl, until all of the mercury present isprecipitated as HgS. The precipitate is then separated, washed and addedas a water suspension to. a 2% solution of cystine -in 0.1 N HCl.

temperature. I After one hour- 75% of the cystine is which may berecovered by conventional procedure.

a Examplelll H S-gas is passed into the reaction mixture at -roomFreshly precipitated Bi S is prepared by passing H 8 aqueous solution ofBi(NO .5 I-I O to by volume of concentrated HNO has been added. Theprecipitate is washed and the aqueous phase displaced by severalwashings with toluene. V 1 part by weight of the toluene-E 8 suspensionis'then added to 10 parts of a 10% solution of 2;2-'dihydrorry6,6-dinaphthyl disulfide:

and H 8 is bubbled through the reaction mixture. After two hours, 70% ofthe disulfide has been converted to the thiol form:

As many changes could be made in the above process and many apparentlywidely different embodiments of a this invention could be made withoutdeparting from the scope of the claims, it is intended that all mattercontained in the above description shall be interpreted as illustrativeand not in a limiting sense.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent of the United States is:

l. The process for reducing disulfide groups in organic disulfidecompounds of the class consisting of cystine, peptides of cystine, andproteins containing cystine, to

'the corresponding sulfhydryl form, in an aqueous medium by treatmentwith hydrogen sulfide in the presence of a 1 sulfide of a-metal selectedfromthe group consisting of mercury, bismuth and lead, at atmosphericpressure and at a temperature below 100 C. e

2."Th'e process defined in'claim 1 in which the molar ratio of r themetal sulfide :to the organicdisulfidexis "between.01 and-1.

below 100 C. and at atmospheric pressure.

proteins containing the same, comprising the treatment of below5.0 inthe 3. The process defined in clairn l in which the molar ratio of themetal sulfide to the organic disulfide is between .01 and 0.1.

4. The process defined in claim 1 in which the aqueous medium has a pHbelow 7.0;

5. The process defined'in claim 1 in which the temperature is maintainedbelow C. V V 7 7 6. The process for reactivating by reduction enzymeproteins inactivated by reason of denaturization to the 8. The processfor reducing cystine by treatmentwith hydrogen sulfide in an aqueousmedium having a pH presence of a sulfide of a metal from the groupconsisting of mercury, bismuth and lead in the molar ratio of .01 to 1.0per mol of the cystine at a temperature below 60 C. and at atmosphericpressure.

9. The process for reducing cystine disulfide bonds in glutathionedisulfide by treatment with hydrogen sulfide in an aqueous medium havinga pH below 5.0. in the presence of a sulfideof a metal from the groupconsisting of mercury, bismuth and lead in the molar ratio of .01 to 1.0per mol of the glutathione disulfide at'a temperature below 60 C. and atatmospheric pressure.

10. A process for reducing cystine disulfide bonds in said proteins inan aqueous medium at a pH below 7.0 with hydrogen sulfide in thepresence of a sulfide of-a metal selected from the group consisting ofmercury, lead and bismuth, in the molar ratio of from 0.1 to 1.0 per molof disulfide present, at a temperature below 60 andat atmosphericpressure.

References Cited in the file of this patent UNITED STATES PATENTS2,051,806 Allen Aug. 25, 1936 2,402,614 Farlow et al. 'June 25, 19462,479,542 Patrick Aug. 16, 1949 2,667,515 Beach et al Jan. 26, 1954OTHER REFERENCES Tarver et a1.: J. Biol. Chem, vol. 146 (1942), pgs.81-2.

1. THE PROCESS FOR REDUCING DISULFIDE GROUPS IN ORGANIC DISULFIDECOMPOUNDS OF THE CLASS CONISTING OF CYSTINE, PEPTIDES OF CYSTINE, ANDPROTEINS CONTAINING CYSTINE, TO THE CORRESPONDING SYLFHYDRYL FORM, N ANAQUEOUS MEDIUM BY TREATMENT WITH HYDROGEN SULFIDE IN THE PRESENCE OF ASULFIDE OF A METAL SELECTED FROM THE GROUP CONSISTING OF MERCURY,BISMUTH AND LEAD, AT ATMOSPHERIC PRESSURE AND AT A TEMPERATURE BELOW100*C.